Anthrax toxin is a major virulence factor of B. anthracis. Anthrax infections are initiated when B. anthracis spores enter a potential host organism by ingestion, inhalation, or skin abrasion. The spores then germinate and replicate as vegetative bacteria, overcome the host innate immune responses, and ultimately enter the circulation leading to a systemic infection. Anthrax toxin consists of the cellular binding moiety protective antigen (PA) and the enzymatic moieties lethal factor (LF) and edema factor (EF). To intoxicate host organisms, PA binds to its cellular receptors TEM8 and CMG2 and is proteolytically activated by the ubiquitously expressed cell surface furin protease, resulting in the formation of active PA oligomer, which in turn translocates LF and EF into the cytosol of cells. LF cleaves several mitogen-activated protein kinase kinases (MEKs), thereby inactivating the ERK, p38, and JUNK MAPK pathways. EF is an adenylate cyclase that generates abnormally high concentrations of cAMP. Development of anthrax toxin vaccines and anti-toxin agents for anthrax prevention and therapy are urgently needed. In the year of 2012, by collaborating with Drs. Schneerson and Robbins (NICHD), we completed a phase 1 study of a recombinant mutant protective antigen (rPA) vaccine in 186 healthy adults. Volunteers were randomized to receive one of three formulations of rPA (formalin treated, alum adsorbed, or both), in 10- or 20-g dosages each, or the licensed vaccine, AVA. Three injections were given at 2-month intervals and a 4th 1 year after the 3rd. We found that all formulations at both dosages were safe and immunogenic, inducing booster responses, with the highest antibody levels following the 4th injection (354 to 732 g/ml). These data are encouraging and support studying alum-adsorbed rPA in children. One effective approach to anthrax therapy is to develop agents that block the actions of anthrax toxin. In the year of 2012, we are continuing collaborative work with Dr. Johnson (PanThera Biopharma, LLC) and have evaluated four small molecule core structures capable of providing sub-nanomolar inhibition of anthrax lethal factor (LF) in a rat lethal toxin (LT) model. We showed that the poor efficacy in the rat LT model exhibited by the phenoxyacetic acid series correlates with their low rat microsome and plasma stability. Specific molecular interactions contributing to the high affinity of inhibitors with a secondary amine in the C2-side chain were revealed by X-ray crystallography. The work provides insights into development of small molecule inhibitors for anthrax lethal factor. We have previously shown that capillary morphogenesis protein-2 (CMG2) is the major anthrax toxin receptor mediating lethality induced by anthrax toxin. However, the physiological functions of CMG2 remain unclear and the consequence of blocking CMG2 activity is largely unknown. To study the roles of CMG2 in normal physiology, in the year of 2012, we performed detailed histological analyses of CMG2-null mice. While no morphological or histological defects were observed in CMG2(-/-) male mice, CMG2(-/-) female mice were unable to produce any offspring due to a defect in parturition. We found that deletion of CMG2 resulted in a diffuse deposition of collagen within the myometrium of CMG2(-/-) females, causing remarkable morphological changes to their uteri. This collagen accumulation also led to loss of smooth muscle cells in the myometrium of CMG2(-/-) mice, apparently disabling uterine contractile function during parturition. As a consequence, even though pregnant CMG2(-/-) mice were able to carry the gestation to full term, they were unable to deliver pups. However, the fully-developed fetuses could be successfully delivered by Cesarean section and survived to adulthood when fostered. Thus, CMG2 is not required for normal mouse embryonic development but is indispensable for murine parturition. One area of work in this project seeks to use modified anthrax toxins to target cancer. Cancer is characterized by the upregulation of a large number of proteolytic enzymes, including urokinase plasminogen activator (uPA). This protease can be expressed by tumor cells, by tumor-supporting stromal cells, or by both. We previously found that the toxicity of anthrax toxin can be redirected to cancers by changing anthrax toxin PAs furin specificity to cancer-selective protease specificities. Therefore, we constructed uPA (urokinase type plasminogen activator)-activated anthrax lethal toxin. In 2012, we explored the use of PA-U2, an engineered anthrax protective antigen that is activated by urokinase, combined with wildtype lethal factor in the treatment of Colo205 colon adenocarcinoma in vitro and B16-BL6 mouse melanoma in vitro and in vivo. This therapy was also tested in combination with the small molecule paclitaxel. Colo205 cells were sensitive to PA-U2/LF while B16-BL6 cells were not. For the combination treatment of B16-BL6, paclitaxel showed a dose response in vitro, but cells remained resistant to PA-U2/LF even in the presence of paclitaxel. In vivo, each therapy slowed tumor progression, and an additive but not synergistic effect between the two was observed. The engineered anthrax toxin PA-U2/LF warrants further development and testing, possibly in combination with an antiangiogenesis therapy such as sunitinib or sorafinib. In addition to being a target for vaccine development, anthrax PA has also been used to deliver passenger polypeptides fused to the N-terminus of LF. Fusion protein 59 (FP59) is such a fusion protein, consisting of LF amino acids 1254 and the catalytic domain of Pseudomonas aeruginosa exotoxin A. FP59 kills cells by ADP-ribosylation of the diphthamide residue on eukaryotic elongation factor 2 (eEF2) after delivery to cytosol by PA. In searching for CHO cell mutants that are resistant to the action of PA + FP59, we found that most of the toxin-resistant mutant cells contain mutations in the diphthamide biosynthesis genes and at least seven such genes have been identified. Interestingly, all the genes required for diphthamide biosynthesis are evolutionally conserved through all eukaryotes from yeast to humans, attesting to the importance of the diphthamide modification on eEF2 in normal physiology. To study the role of the diphthamide modification on eEF2, we generated an eEF2 Gly(717)Arg mutant mouse, in which the first step of diphthamide biosynthesis is prevented. Interestingly, the Gly(717)-to-Arg mutation partially compensates the eEF2 functional loss resulting from diphthamide deficiency, possibly because the added +1 charge compensates for the loss of the +1 charge on diphthamide. Therefore, in contrast to mouse embryonic fibroblasts (MEFs) from OVCA1(-/-) mice, eEF2(G717R/G717R) MEFs retain full activity in polypeptide elongation and have normal growth rates. Furthermore, eEF2(G717R/G717R) mice showed milder phenotypes than OVCA1(-/-) mice (which are 100% embryonic lethal), and a small fraction survived to adulthood without obvious abnormalities. Moreover, eEF2(G717R/G717R)/OVCA1(-/-) double mutant mice displayed the milder phenotypes of the eEF2(G717R/G717R) mice, suggesting that the embryonic lethality of OVCA1(-/-) mice is due to diphthamide deficiency. We confirmed that the diphthamide modification is essential for eEF2 to prevent -1 frameshifting during translation and show that the Gly(717)-to-Arg mutation cannot rescue this defect. This work demonstrates that the diphthamide modification on eEF2 is needed to assure fidelity of mRNA translation and mouse development.